A highly conserved mycobacterial cholesterol catabolic pathway

Area of Science:

• Biochemistry
• Microbiology
• Structural Biology

Background:

• Mycobacterium tuberculosis initiates cholesterol side-chain degradation using cytochromes P450 CYP125A1 and CYP142A1.
• Cholesterol is a vital nutrient for M. tuberculosis, and its degradation pathway is a potential drug target.

Purpose of the Study:

• To characterize the homologous enzymes CYP125A3 and CYP142A2 from Mycobacterium smegmatis.
• To investigate the substrate binding and catalytic activity of these enzymes.
• To elucidate the functional redundancy in cholesterol degradation within M. smegmatis.

Main Methods:

• Heterologous expression and purification of CYP125A3 and CYP142A2.
• Enzyme activity assays using cholesterol derivatives and reconstituted electron transfer systems.
• X-ray crystallography to determine the structures of substrate-free and ligand-bound enzymes.
• Gene deletion studies to assess the in vivo function and redundancy of the enzymes.

Main Results:

• CYP125A3 and CYP142A2 were successfully expressed, purified, and shown to bind cholesterol, 4-cholesten-3-one, and azole drugs.
• Both enzymes efficiently hydroxylated 4-cholesten-3-one to alcohol and acid metabolites.
• X-ray structures revealed distinct substrate binding sites compared to M. tuberculosis homologs.
• Deletion of cyp125A3 led to increased cyp142 expression, indicating functional redundancy.
• A double mutant lacking both genes still retained cholesterol growth capability, suggesting further genomic redundancy.

Conclusions:

• CYP125A3 and CYP142A2 are key enzymes in M. smegmatis cholesterol metabolism.
• CYP142A2 provides functional backup for CYP125A3 in cholesterol degradation.
• M. smegmatis possesses a higher degree of redundancy in cholesterol metabolism compared to M. tuberculosis.